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Abstract

Slc26a4 (Pds, pendrin) is an anion transporter expressed in the apical region of type B and non-A, non-B intercalated cells of the distal nephron. It is upregulated by aldosterone analogues and is critical in the development of mineralocorticoid-induced hypertension. Thus, Slc26a4 expression and its role in blood pressure and fluid and electrolyte homeostasis was explored during NaCl restriction, a treatment model in which aldosterone is appropriately increased. Ultrastructural immunolocalization, balance studies, and cortical collecting ducts (CCDs) perfused in vitro were used. With moderate physiological NaCl restriction, Slc26a4 expression in the apical plasma membrane increased 2- to 3-fold in type B intercalated cells. Because Slc26a4 transports Cl−, we tested whether NaCl balance differs in Slc26a4(+/+) and Slc26a4(−/−) mice during NaCl restriction. Cl− absorption was observed in CCDs from Slc26a4(+/+) but not from Slc26a4(−/−) mice. After moderate NaCl restriction, urinary volume and Cl− excretion were increased in Slc26a4(−/−) relative to Slc26a4(+/+) mice. Moreover, Slc26a4(−/−) mice had evidence of relative vascular volume depletion because they had a higher arterial pH, hematocrit, and blood urea nitrogen than wild-type mice. With moderate NaCl restriction, blood pressure was similar in Slc26a4(+/+) and Slc26a4(−/−) mice. However, on a severely restricted intake of NaCl, Slc26a4(−/−) mice were hypotensive relative to wild-type mice. We conclude that Slc26a4 is upregulated with NaCl restriction and is critical in the maintenance of acid-base balance and in the renal conservation of Cl− and water during NaCl restriction.

In the kidney, intercalated cells secrete net acid or base depending on whether the H+-ATPase localizes to the apical or the basolateral plasma membrane1 and whether the cell expresses anion transporters such as AE1 and Slc26a4.2–5 Although the physiological role of non-A, non-B intercalated cells is unknown,3 type B intercalated cells are a critical component of the robust ability of the kidney to excrete OH− equivalents during metabolic alkalosis.4 In this cell type, the apical plasma membrane secretes HCO3− into the luminal fluid, through Cl−/HCO3− exchange, in series with H+- efflux across the basolateral plasma membrane.2,3,6

Slc26a4 localizes to the apical plasma membrane and apical cytoplasmic vesicles of type B and non-A, non-B intercalated cells within the cortical collecting duct (CCD), the connecting tubule (CNT), the initial collecting tubule (iCT), and the distal convoluted tubule (DCT).5,7,8 Because Slc26a4 transports HCO3− and participates in bicarbonate secretion by type B intercalated cells,5,9 previous studies have focused on the role of Slc26a4 in acid-base balance and have shown that Slc26a4 is upregulated in models of metabolic alkalosis. With administration of aldosterone analogues (deoxycorticosterone [DOCP]), Slc26a4 expression is upregulated in tandem with upregulation of apical Cl−/HCO3− exchange, which increases HCO3− secretion.10,11 After administration of DOCP, Slc26a4(−/−) mice fail to secrete HCO3− in the CCD and develop a more severe metabolic alkalosis than wild-type mice.5,11 Thus, Slc26a4-mediated HCO3− secretion attenuates the metabolic alkalosis observed in this treatment model.

In addition to secretion or absorption of H+/OH− equivalents, intercalated cells also participate in the secretion or absorption of Cl− through transcellular transport.12–14 Across intercalated cells, aldosterone stimulates Cl− absorption through apical Cl−/HCO3− exchange in series with a basolateral Cl− conductance.14 Thus, it is likely that aldosterone analogues activate Slc26a4-mediated apical Cl− uptake in type B and in non-A, non-B intercalated cells.

Within the aldosterone-sensitive region of the nephron, NaCl absorption is greatest in those segments where Slc26a4 expression is highest (ie, the CNT and the CCD).7,8,15 We have made 2 observations that suggest that the increase in Cl− absorption by the CCD and CNT following administration of aldosterone analogues is Slc26a4-dependent.11 First, Slc26a4 is greatly upregulated with DOCP. Second, the weight gain and hypertension expected in wild-type mice after administration of aldosterone analogues is not observed in Slc26a4 null mice.11 However, no direct link between Slc26a4 and the renal absorption of NaCl and water has been demonstrated.

With reduced NaCl intake, the renin-aldosterone-angiotensin system is activated, which augments vascular tone and renal NaCl absorption. However, the role of direct activation of renal Cl− transporters such as Slc26a4 to conserve NaCl and water, and hence to maintain vascular volume, is unknown. Therefore, we asked 3 questions: First, does physiological variation in NaCl intake modulate Slc26a4 expression? Second, does NaCl restriction unmask a difference between Slc26a4(+/+) and Slc26a4(−/−) mice in renal NaCl and water balance? And third, does Slc26a4 modulate transepithelial Cl− transport in renal segments that highly express the transporter?

Methods

Animals

For series 1, male, non-Swiss albino mice (Harlan) weighing 20 to 30 g were divided into 2 groups at random. One group received a balanced, NaCl-restricted diet (0.13 mEq per day NaCl; 53881300; Zeigler Brothers) for 7 days prepared as a gel (0.6% agar, 74.6% water, and 24.8% mouse chow). The other group was pair fed the same gel diet but supplemented with NaCl (NaCl-replete diet; 0.78 mEq per day NaCl). For series 2, Slc26a4(−/−) mice developed by Everett et al16 were bred in parallel with coisogenic wild-type mice (129S6/SvEv Tac; Taconic Farms). For 7 days before euthanization, age- and sex-matched Slc26a4(+/+) and Slc26a4(−/−) mice were pair fed the gel diet with or without NaCl supplementation (0.13 or 0.78 mEq per day; NaCl). For series 3, Slc26a4(−/−) and Slc26a4(+/+) mice were pair fed a NaCl-deficient diet (<0.01 mEq per day; NaCl; 53140000; Zeigler Brothers) prepared as a gel. Series 1 to 3 mice were placed in metabolic cages, and urine was collected under oil at 4°C for 24 hours before euthanization. Mice in series 1 to 3 were euthanized under anesthesia with 1 to 4% isofluorane in 100% O2 at 1 L/min. For series 4, Slc26a4(−/−) and Slc26a4(+/+) mice ate a balanced rodent diet (LabDiet 5001; PMI Nutrition International) and were given 5 mg/100 g body weight DOCP pivalate (Novartis) by intramuscular injection and drank water containing 50 mmol/L NaHCO3 ad libitum for 5 to 9 days before euthanization.5 The institutional animal care and use committee at Emory approved all animal treatment protocols.

Systolic blood pressure in conscious mice was measured by tail cuff using a BP-2000 (Visitech Systems). Blood was collected for serum chemistries through the abdominal aorta under isofluorane anesthesia. Urine Na+ from mice on a low-salt diet was measured by atomic emission spectroscopy in a Perkin-Elmer AA model 3110 with an HGA-600 graphite furnace with standards and samples diluted with deionized glass-distilled water. Chloride concentration in urine from mice on a low-salt diet was determined by coulombometric titration with a CMT 10 chloride titrator (Radiometer). Otherwise, serum and urine chemistries, urinary pH, and urinary excretion of osmoles, ammonium, and net acid were measured as reported previously.11 Arterial blood gases were measured as described previously11 using an OPTI 1 blood gas analyzer (AVL Medical Instruments). Serum aldosterone was measured using a Coat-A-Count aldosterone radioimmunoassay (RIA) kit (Diagnostic Products). Free corticosterone in urine was measured by double-antibody RIA (item I, available online at http://www.hypertensionaha.org) with materials from MP Biomedicals (ImmuchemCorticosterone).

Preparation of Total RNA and Quantitative Real-Time RT-PCR

Total RNA was isolated from mouse kidney as reported previously.7 Quantitative real-time PCR was performed in the Quantitative Genomics Core Laboratory in the Department of Integrative Biology and Pharmacology, University of Texas, Medical School at Houston (UTHMC), using specific quantitative assays for mouse Slc26a4 and β-actin mRNA, as reported previously.7

Immunogold Cytochemistry

Kidneys were prepared for electron microscopy as described previously.7 For electron microscopy, Slc26a4 immunoreactivity was localized in ultrathin sections using immunogold cytochemistry.7 The CCD, connecting segments (CNT), and iCTs were identified as described previously.7 Type A, type B, and non-A, non-B intercalated cell subtypes were identified using morphological characteristics established in studies of rat and mouse under basal conditions.7

Morphometric Analysis

Apical plasma membrane boundary length, cytoplasmic area, and gold label along the apical plasma membrane and over the cytoplasm, including cytoplasmic vesicles, were quantified in type B and non-A, non-B intercalated cells from each treatment group as described previously.7 In each animal, ≥5 cells of each intercalated cell subtype were selected at random and photographed at a primary magnification of ×5000 and examined at a final magnification of approximately ×18 200.

Statistical Analysis

For morphometric data without normal distribution or equal variance, a Mann-Whitney rank sum test was used. In all other studies, comparisons were made between 2 groups using an unpaired Student t test. P<0.05 indicates statistical significance. Data are displayed±SEM.

We asked whether Slc26a4 expression increases with moderate NaCl restriction, a model in which serum aldosterone is appropriately increased. Slc26a4 and β-actin mRNA/kidney were similar in kidneys from NaCl-replete and NaCl-restricted mice (series 1; Table 1). Therefore, modest NaCl restriction does not significantly change Slc26a4 message expression in kidney.

Effect of NaCl Restriction on the Expression and Subcellular Distribution of Slc26a4

Because aldosterone analogues induce a marked change in the subcellular distribution of Slc26a4,11 we tested the effect of moderate NaCl restriction on the magnitude and subcellular distribution of Slc26a4 protein expression (Figure; Table 2; items II and III, available online at http://www.hypertensionaha.org). Slc26a4 expression in the apical plasma membrane and cytoplasm was quantified in type B and non-A, non-B intercalated cells from NaCl-replete and NaCl-restricted mice (series 1). In NaCl-replete animals, the ultrastructural features and distribution of Slc26a4 immunolabel were similar to our previous observations.7 Type B cells have a smooth apical plasma membrane surface, numerous cytoplasmic vesicles, a subapical band free of vesicles, and abundant mitochondria (Figure, a).7Slc26a4 immunoreactivity was prevalent over apical cytoplasmic vesicles, but little immunolabel was present along the apical plasma membrane (Figure, a). NaCl restriction led to a 2- to 3-fold increase in Slc26a4 label along the apical plasma membrane (Figure, b) because of increased label density rather than changes in boundary length. Moreover, the ratio of immunogold label in the apical plasma membrane to label in the cytoplasm and cytoplasmic vesicles increased 2.4-fold. However, in non-A, non-B intercalated cells, Slc26a4 expression in the apical plasma membrane did not change with NaCl restriction. Moreover, NaCl restriction did not change total cell Slc26a4 expression in either of these intercalated cell subtypes. We conclude that NaCl restriction induces a shift in the subcellular distribution of Slc26a4 in the type B intercalated cell of the CCD, resulting in increased expression of Slc26a4 in the apical plasma membrane, with little change in total Slc26a4 protein expression per cell.

Effect of NaCl Restriction on Slc26a4(+/+) and Slc26a4(−/−)Mice

Because Slc26a4 is upregulated with NaCl restriction, further studies asked whether a renal phenotype is unmasked in Slc26a4(−/−) mice after NaCl restriction. Because Slc26a4 transports HCO3−,9 arterial blood gases were measured in Slc26a4(−/−) and Slc26a4(+/+) mice after an NaCl-restricted or an NaCl-replete diet. As reported previously,11 after an NaCl-replete diet, arterial pH was similar in Slc26a4(−/−) and Slc26a4(+/+) mice (series 2; Table 3). However, Slc26a4(−/−) mice but not wild-type mice developed metabolic alkalosis during moderate NaCl restriction (Table 3). Slc26a4(−/−) mice had increased excretion of titratable acid after NaCl restriction (Table 3), although no difference in net acid excretion was detected between Slc26a4(+/+) and Slc26a4(−/−) mice. We conclude that Slc26a4 expression attenuates the metabolic alkalosis that can occur during NaCl restriction.

TABLE 3. Serum and Urine Chemistries in Slc26a4(−/−) and Slc26a4(+/+) Mice on an NaCl-Replete or Restricted Diet (Series 2)

Table 3 shows that on an NaCl-replete diet, serum electrolytes, urinary volume, and NaCl excretion were the same in Slc26a4(−/−) and Slc26a4(+/+) mice. After 7 days of the NaCl-restricted diet (series 2), serum electrolytes were also similar in wild-type and Slc26a4(−/−) mice. However, after moderate NaCl restriction, Slc26a4(−/−) mice had greater excretion of Cl− and greater urinary volume than Slc26a4(+/+) mice. Although Mg2+ excretion was slightly increased in Slc26a(−/−) mice, no difference was detected between knockout and wild-type mice in excretion of other cations (ie, Na+, K+, NH4+, and Ca2+; Table 3; item IV, available online at http://www.hypertensionaha.org).

Because of the observed chloruresis, we asked whether Slc26a4(−/−) mice are more volume contracted than wild-type mice during NaCl restriction. Table 3 demonstrates that on an NaCl-replete diet, blood urea nitrogen (BUN) and hematocrit were the same in Slc26a4(−/−) and Slc26a4(+/+) mice. However, on an NaCl-restricted diet, BUN and hematocrit were higher in Slc26a4(−/−) than Slc26a4(+/+) mice. Thus, Slc26a4(−/−) mice show apparent vascular volume contraction relative to wild-type mice during NaCl restriction, as expected after increased excretion of Cl− and water. However, with moderate NaCl-restriction (series 2; Table 4), Slc26a4(−/−) mice maintain blood pressure, despite an apparent fall in vascular volume. Therefore, we asked whether Slc26a4(−/−) mice have a reduced ability to maintain blood pressure with further NaCl restriction. After an NaCl-deficient diet (series 3), systolic blood pressure was lower in Slc26a4(−/−) than in Slc26a4(+/+) mice (Table 4). We conclude that Slc26a4(−/−) mice have a compromised ability to maintain blood pressure after severe NaCl restriction.

Role of Slc26a4 in Cl− Absorption in the CCD

To examine the role of Slc26a4 in Cl− absorption in the CCD, Slc26a4(−/−) and Slc26a4(+/+) mice were given DOCP and drank water containing NaHCO3 (series 4) to upregulate Slc26a4 expression.11,18 CCD tubules from Slc26a4(−/−) and Slc26a4(+/+) mice were perfused in vitro and JCl was measured in each group. JCl was +4.7±1.4 (n=6) pmol/mm per minute in Slc26a4(+/+) mice and −2.0±1.5 pmol/mm per minute (n=4; P<0.05) in Slc26a4(−/−) mice. Thus, Cl− absorption was detected in the CCD of wild-type mice but not in CCDs from Slc26a4(−/−) mice (item V, available online at http://www.hypertensionaha.org). This defect in Cl− absorption contributes to the chloruresis observed in NaCl-restricted Slc26a4(−/−) mice.

Discussion

The human body has a robust ability to maintain vascular volume and blood pressure after NaCl depletion. Normal adults can lose up to 30% of their total body Na+ and Cl− without changes in blood pressure.19 After NaCl restriction, vascular volume and hence blood pressure are maintained through multiple mechanisms that include the release of renin, angiotensin II, aldosterone, and catecholamines. The result is increased absorption of NaCl by the kidney, colon, and sweat glands, and increased vascular tone.20–24

The renin/angiotensin/aldosterone axis is critical to the regulation of net acid secretion and absorption of NaCl along the collecting duct. Limited information is available as to the effect of angiotensin II on Cl− transport in the CCD. However, it is well established that aldosterone administration markedly increases secretion of HCO3− and K+ and increases absorption of Na+ and Cl− in the CCD, which contributes to the maintenance of vascular and interstitial volume. Whereas the effect of aldosterone on Na+ absorption in the kidney has been well studied, the mechanism of aldosterone-induced Cl− absorption is poorly understood.

Increased Slc26a4-mediated Cl− absorption contributes to the reduction in urinary Cl− excretion during NaCl restriction. Thus, genetic disruption of Slc26a4 produces an inappropriate chloruresis and apparent vascular volume contraction during NaCl restriction. The inability of Slc26a4(−/−) mice to conserve Cl− leads to a reduced ability to maintain blood pressure during NaCl restriction. Conversely, the absence of hypertension in Slc26a4 knockout mice after DOCP administration likely occurs, at least in part, from the inability of the DCT, CNT, and CCD of Slc26a4(−/−) mice to increase Cl− absorption fully in response to aldosterone analogues. However, the signaling mechanism of aldosterone analogue-induced activation of Slc26a4 is unresolved (item VI, available online at http://www.hypertensionaha.org). Moreover, we cannot exclude the possibility that genetic disruption of Slc26a4 changes glomerular filtration rate or changes expression of other Cl− transporters, which may contribute to the Cl− wasting observed in the present study (item VII, available online at http://www.hypertensionaha.org). Finally, which cation(s) follows the increase in Cl− excretion in Slc26a4(−/−) mice during NaCl restriction is unclear because differences between Slc26a4(−/−) and Slc26a4(+/+) mice with regard to Na+, K+ NH4+, Mg2+, or Ca2+ excretion were either much smaller than differences in Cl−excretion or not statistically significant.

Changes in Slc26a4 expression have been studied in various in vivo models of metabolic alkalosis.11,18,25 These models induce complex physiological changes, including changes in the renin, angiotensin, serum K+, etc, any of which might directly alter Slc26a4 expression. However, these studies collectively demonstrate certain patterns with regard to regulation of Slc26a4 expression. In many treatment models studied, Slc26a4 expression changes in tandem with expected changes in serum aldosterone. Na+ restriction increases serum aldosterone,26 which most probably increases Slc26a4 expression.11 However, during NaCl restriction, changes in dietary Cl− intake, which may not change serum aldosterone,26 might regulate Slc26a4 expression. For example, Slc26a4 expression is upregulated when dietary NaCl is substituted with NaHCO3.18 Thus, upregulation of Slc26a4 during NaCl restriction could occur from reduced intake of Na+ or Cl−.

Slc26a4(−/−) mice but not wild-type mice develop metabolic alkalosis during moderate NaCl restriction. Thus, Slc26a4 is required for maintenance of acid-base balance in this treatment model, likely by mediating HCO3− secretion in the CCD and CNT. Slc26a4, studied in heterologous expression systems, and the apical anion exchanger of the type B intercalated cell, studied in native tissue, transport Cl− and HCO3− as Cl−/Cl and Cl−/HCO3− exchange.4,9,14,27,28 The present and previous studies demonstrate that Slc26a4 might act as an electroneutral Cl−/HCO3− exchanger in native kidney tissue because Cl− absorption and HCO3− secretion observed in CCDs from Slc26a4(+/+) mice is not observed in CCDs from Slc26a4(−/−) mice. However, we cannot exclude the possibility that Slc26a4 functions as a Cl− transporter and indirectly affects HCO3− transport through regulation of other H+/OH− transport processes within the CCD. For example, the absence of Pds-mediated Cl− absorption may increase distal delivery of Cl−, leading to increased secretion of H+ equivalents. Clarification of this mechanism will require further studies in native tissue.

Humans with genetic disruption of Slc26a4 (Pendred Syndrome) have no known symptoms attributable to renal abnormalities, at least under basal conditions. Whether abnormalities in blood pressure or abnormalities in acid-base or fluid and electrolyte balance become apparent in these patients under conditions that upregulate Slc26a4 in normal individuals remains to be determined. Patients with Pendred Syndrome develop deafness from structural malformations of the inner ear and endolymphatic swelling (endolymphatic hydrops), which may occur from reduced Cl− absorption within the inner ear,16 as observed in kidney.

In conclusion, Slc26a4 is upregulated during NaCl restriction. With changes in NaCl intake, Slc26a4 expression in the apical plasma membrane changes more in the type B than in the non-A, non-B intercalated cell. Slc26a4 is critical in the renal conservation of Cl− during NaCl restriction and contributes to the maintenance of arterial pH during NaCl restriction, either through direct Pds-mediated transport of OH− equivalents or through indirect effects such as changes in distal delivery of Cl−.

Perspectives

The present study is the first to demonstrate a role of Slc26a4 in the renal conservation of NaCl and raises the possibility that chemical inhibitors of Slc26a4 will act as diuretics.

Footnotes

↵1Eating 4.5 g per day of the standard rodent diet used at the Emory Department of Animal Resources (5001 LabDiet; PMI Nutrition International), mice will consume 0.78 mEq per day Na+ and 0.82 mEq per day Cl−. Intake of 0.13 mEq per day NaCl exceeds the minimum nutrient requirement for mice.17